The invention generally relates to compositions and methods for treating disorders related to tyrosinase gene expression and melanin biosynthesis. In addition, the invention relates to a model for pigmentation of hair.
Treatment of the hair and skin with various creams or lotions with biologically active ingredients to improve hair growth and pigmentation has generally been unsatisfactory. A wide variety of externally applied agents available are said to improve body, flexibility, curl and hair color. These have limited and only short term usefulness. In particular, coloring hair with various dyes requires frequent repetitions and is not always natural in appearance. The invention provides improved alternatives, focused on tyrosinase.
Tyrosinase is a ubiquitously distributed copper-containing monoxygenase that is essential for melanin biosynthesis in pigment cells. It catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA) and the conversion of DOPA to dopaquinone, referred to as tyrosine hydroxylase activity and DOPA oxidase activity, respectively.
Disorders of tyrosinase gene expression and melanin biosynthesis are related to many diseases involving pigmentation such as albinism, hair pigment loss, and vitellego. Tyrosinase is a key enzyme for melanin synthesis in vertebrate pigment cells, melanocytes, and retinal pigment epithelial cells. Tyrosinase is absent in human white hair bulbs, as well as in albino epithelial cells. Thus, the loss of tyrosinase could be the basis of pigment loss in hair. It is also believed that tyrosinase activity is abnormal in Parkinson""s disease. Tyrosinase is expressed in brain cells in the substantia nigra, forebrain, and midbrain, thus tyrosinase could be implicated in neuromelanin formation in the substantia nigra and thus related to neurodegenerative disorders.
The murine and human tyrosinase genes have been cloned. (Kwon et al. Proc Natl Acad Sci USA (1987) 84:7473-7477; Yamamoto et al. Jpn J Genet (1989) 64:121-135; Ruppert etal. EMBO J (1988) 7:2715-2722. The structural genes for human and murine tyrosinase have long DNA sequences. In mice, the structural gene for tyrosinase is located on the c-locus of chromosome 7, which spans a region of more than 70 kb. In humans, the tyrosinase gene is located on the c-locus on chromosome 11. It has been reported that only the full complement of five exons were able to confer tyrosinase enzyme activity to tyrosinase-negative cells.
Murine and human tyrosinase cDNAs have been expressed in mammalian cells after transfection. See Muller et al. (1988), Tomita et al. (1989), Yamamoto et al. (1989) supra, Geibal et al. (1990), Porter et al. Gene (1991) 97:277-282; Mishima et al. (1993), and U.S. Pat. No. 5,753,263. Several cDNA clones of the mouse tyrosinase have been isolated independently. Among those cDNA clones, there are several differences in the deduced amino acid sequence. Muller et al. (1988) supra reported that one of their cDNA clones, pmcTYr1, possessed the coding capacity for tyrosinase, and they demonstrated that pmcTYr1 could be inserted in cultured human amelanotic melanoma cells. However, they did not report any pigment production. On the other hand, Yamamoto et al. (1989) supra constructed the mouse tyrosinase cDNA minigene, mg-Tyr1-J which was ligated with a genomic 5xe2x80x2-non-coding flanking sequence and transfected cultured albino melanocytes, which produced tyrosinase and melanin.
The tyrosinase gene in albino mice has a single base-pair mutation in exon 1 which results in a nonfunctional tyrosinase protein and absence of melanin in the albino melanocytes. Melanin has been produced in amelanotic melanocytes in transgenic albino mice whose fertilized eggs were inserted with a normal mouse tyrosinase transgene, and melanin was seen in the hair bulbs, hair shafts, choroid, and pigment epithelium. Tanaka et al. Development (1990) 108:223-227; Beerman et al. Pigment Cell Research (1992) 5:295-299. Human tyrosinase cDNA has been used to transfect amelanotic melanoma cells and induce melanin production. Ando et al. J Invest Dermatol (1993) 101:864-870.
The human and murine tyrosinase constructs are difficult to manipulate due to their complexity and size. Many species of Streptomyces are also capable of forming melanin due to the expression of tyrosinase from the mel operon. See Kuster et al. (1976) Chromogenicity of actinomycetes. Actinomycetes: The Boundary of Microorganisms. Ed. T. Arai, Tokyo: Toppan Co. 43-45. The 1.2 kb mel locus of S. antibioticus has been cloned and sequenced and shown to contain a structural tyrosinase gene and an open reading frame ORF-438. The ORF-438 protein regulates copper incorporation, which is essential for the expression and function of tyrosinase and melanin production in Streptomyces. The ORF-438 and tyrosinase genes are transcribed from the same promoter located in the 5xe2x80x2-region adjacent to ORF438, thereby indicating that these genes form an operon. Beman et al. Gene (1985) 37:101-110. The ORF-438 protein functions as a transactivator of tyrosinase. See Lee et al. Gene (1988) 65:71-81. The ORF-438 protein also delivers copper to apotyrosinase to generate active tyrosinase. Production of melanin in E. coli from the mel locus of S. antibioticus was dependent on the coexpression of a full length ORF-438 gene as well as the tyrosinase gene. See Della-Cioppa et al. Biotechnology (1990) 8:634-638.
Advantage has been taken of the characteristics of the mel operon which is smaller in size, lacks introns, and exhibits a potential for high expression to construct vectors useful to construct models for pigmentation disorders and for treatment of tyrosinase deficiency.
Generally, the present invention is directed to compositions and methods for treating disorders related to tyrosinase gene expression and melanin biosynthesis, and to model systems for such disorders.
In one aspect, the invention is directed to a nucleic acid molecule comprising a bicistronic nucleotide sequence for efficient production of a functional Streptomyces tyrosinase in mammalian cells comprising a tyrosinase encoding nucleotide sequence and nucleotide sequence encoding ORF-438, coupled through an internal ribosome entry sequence (IRES).
In another aspect, the invention is directed to a method for treating a tyrosinase deficiency or pigmentation disorder in a subject which method comprises modifying the cells of the subject with the expression system of the invention. In a preferred embodiment, the cells to be treated are hair follicle cells and the subjects are mammals, such as primates, including humans, domestic animals, and rodents. The expression system can be utilized as the sole method of treatment, but the system may be used in combination with administration of other beneficial compounds such as proteins, pigments, dyes, growth regulators and other compounds which affect the characteristics of skin and hair. Thus, the expression system may be utilized in addition to cell cycle regulating proteins or the multi-drug resistance proteins which confer resistance to chemotherapy-induced alopecia.
In another aspect, the invention is directed to cells modified to contain the expression system of the invention for the expression of active tyrosinase and production of melanin. In another aspect, the invention is directed to a nonhuman animal comprising the modified cells. The invention is also directed to vectors which comprise the nucleotide sequence encoding tyrosinase and a protein for the incorporation of copper.
In another embodiment, the invention is directed to a method to monitor the effect of compounds or protocols on pigment disorders using histocultures of cells containing the expression systems of the invention.